**2.2 Diabetic retinopathy**

Diabetic retinopathy is a medical condition where damage to retina, as a result of high glucose, occurs. It is the most frequent cause of blindness in patients with diabetes. Patients with DR usually does not develop any major symptoms at an early stage but during later stages physiological and metabolic abnormalities can appear leading to blindness, if left untreated. The risk factors associated with DR includes high blood glucose [20], duration and type of diabetes [21], high B.P. [22] and, lipids [23]. Presently it is being diagnosed with the identification of microvascular lesions in the retina. It has been differentiated clinically in 2 categories on the basis of ophthalmic observation: proliferative DR (PDR), the advance stage and; non-proliferative DR (NPDR), the early stage. NPDR can be identified by fundus where hard exudates, microaneurysms or hemorrhages are seen. NPDR is further categorized into mild, moderate and severe NPDR. On the other hand, detection of retinal neovascularization confirms PDR. The risk of progression of DR can

*Type 2 Diabetes - From Pathophysiology to Cyber Systems*

of adult population in the world [3].

[5], which are the main vascular complications of diabetes.

**2. Diabetes mellitus-associated vascular complications**

diabetes instigates vascular diseases affecting almost all blood vessels of the body, which further results in increased morbidity and mortality in diabetic populations. Among well-known risk factors of diabetes, non-changeable factors include genetics, age and ethnicity while others are changeable, for example physical activity, adiposity, environmental exposures and diet, via combination of treatment at both individual as well as population level [3]. Type 2 diabetes is frequently seen in older adults, but now-a-days, may be, as a result of increasing physical inactivity, obesity and/or the absence of healthy diet it is also being seen increasingly among children, teenagers and younger adults. Diabetes is globally affecting 425 million people or 8.8% of adult population. By 2045, diabetes is projected to affect about 629 million

India, now-a-days, is becoming the diabetes capital of the world with estimated prevalence of diabetes as 7.3% and that of pre-diabetes as 10.3% [4]. In the current report (2017), 72.9 million Indians were suffering from diabetes and this is expected to rise to 134.3 million by the year 2045 [3]. Prolonged hyperglycemia is the foremost cause of kidney disease, cardiovascular disorders, retinopathy and neuropathy

Hyperglycemia triggers damage to the vasculature and thus, leads to the failure of various organs including kidney, heart, retina of eyes and nerves; usually develop after many years of diabetes. This gives rise to the development of vascular complications which are categorized into micro- and macrovascular complications. Microvascular disease or microangiopathy is actually the thickening of walls of small blood vessels so that they bleed and leakage of protein occurs. This narrowing of blood vessels results in decreased blood flow and impairment of oxygen flow throughout the body which leads to the damage of tissues or organs that are extremely sensitive to oxygen levels i.e., kidney cells, nerve cells and retina. On the other hand, macrovascular disease or macroangiopathy is the disease of large blood vessels due to clot formations that further results in the decreased blood flow all through the body. This may cause heart diseases, peripheral vascular diseases or stroke. Both micro- and macrovascular complications are the result of hyperglycemia and it seems that they both may be interconnected but who precedes whom or whether they progress together, it is not clear. Complications of T2DM keep on increasing due to increasing burden of diabetes, thus deteriorating the quality of human life. Smoking, age factor, increased weight, lack of physical activity and high-fat diet are the common risk factors to diabetes complications. Now-a-days diabetic kidney disease (DKD) or DN and diabetic retinopathy (DR) are among the most frequent complications of diabetes. Improved and maintained glycemic control may reduce risk of some of the diabetic complications, but it is not the only factor which, if under control, may reduce the progression of all vascular complications. In this segment, we have elaborated two major microvascular complications

Diabetic nephropathy is the major microvascular complication of diabetes affecting 20–30% of patients with type 2 diabetes mellitus [6], which weaken the quality of life leading to increased morbidity and mortality. Symptoms of DN are less evident in the early years of diabetes, usually develops after many years of diabetes. In India approximately 48% cases of CKD are caused by diabetes [7].

**172**

of diabetes, i.e., DN and DR.

**2.1 Diabetic nephropathy**

be reduced by early detection, but it is difficult to achieve as there is little or no symptoms at early stages. Several molecular mechanisms are thought to involve in the development and progression of DR including polyol pathway, enhanced expression of vascular endothelial growth factor (VEGF), production of advance glycation end products (AGEs), activation of RAAS, hemodynamic alterations, etc. Current treatment involves conventional laser therapy and anti-VEGF or other antiangiogenic, anti-inflammatory, non-steroidal anti-inflammatory drugs (NSAIDs) treatment. Despite this, reading is also difficult in patients with severe retina loss. Some treatments are precise but they are associated with high cost or side effects. Hence, the discovery of fundamental molecular mechanisms involved is required for the development of more specific interventions. Among genetic predisposition to the disease, several candidate genes have been identified in the past few decades with contradictory findings, although few genes have been found to be associated with DR in mostly studies. Among them, aldose reductase (AKR1B1) is important enzyme in polyol pathway. Activation of this pathway and AKR1B1 polymorphisms are incriminated in the pathogenesis of DR [24]. VEGF is another the most important growth factor activated by hyperglycemia and implicated in the development and progression of DR [25]. Various polymorphisms of VEGF were found to be associated with DR with conflicting results [24]. ACE I/D polymorphism was found to be associated with PDR in a meta-analysis [26]. Receptor for advance glycation end products (RAGE) gene polymorphisms is also reported to be associated with DR in Indian population [27].

The pathophysiology of complications of diabetes is very complex as depicted in **Figure 1**.

#### **Figure 1.**

*Signaling pathways facilitating the pathogenesis of microvascular complications of diabetes mellitus.*

**175**

*Diabetes Microvascular Complications: An Overview of Epigenetic Modifications*

*Diabetes-induced hyperglycemia promotes various growth factors which play influential roles in the progression of diabetic complications. These factors act by binding to their specific receptors to initiate multiple downstream signaling cascades involved. Subsequently, these signaling pathways trigger transcription factors and promote their crosstalk with epigenetic mechanisms that lead to diabetic microvascular complications. Transcription factors also interact with epigenetic factors that further participate in metabolic memory. AT1R, Angiotensin II type 1 receptor; AGEs, Advance glycation end products; TGF-β, Transforming growth factor-β1; ROS, Reactive oxygen species; NO, Nitric oxide; Akt, Serine/threonine-specific protein kinase; PKC, Protein kinase C; MAPK, Mitogen-activated protein kinase; NF-κB, Nuclear factor-κB; USFs, Upstream* 

Several genetic factors and gene polymorphisms have been extensively discovered, studied and implicated in DN as well as DR, but no report is able to provide strong evidence regarding uneven response to available treatment. No drug or treatment is able to provide a stable and long-term protective effect against these

In past few years, a lot of interest has been generated in gene–environment interactions as they seem to be involved in the pathophysiology of diabetes mellitus. Recently, epigenetic mechanisms have been linked to various complications of diabetes, as altered gene expressions are the results of several post-transcriptional modifications (PTMs) of chromatin. Accomplishing complete control of blood glucose is also not sufficient to stop or retard the development and progression of diabetes complications; this proposes the involvement of initial glycemic 'metabolic memory' in various complications of diabetes. So, how epigenetic modifications play the role in diabetic vascular complications is still not completely understood, therefore, we have described the understanding about epigenetic mechanisms and

their role in the pathophysiology of diabetic microvascular complications.

In mammalian cells, expression of a gene is known to be controlled by genetic as well as epigenetic mechanisms. In the recent times, epigenetic mechanisms have been shown to play substantial roles in the development and progression of diabetes and its microvascular complications. In this section, epigenetic mechanisms have

Epigenetic mechanisms are deprived of any modification in the principal DNA

structure which involves vibrant switching within 'active' (euchromatin) and 'inactive' (heterochromatin) positions of chromatin; that determined 'gene activation' and 'gene repression' states and thus biological outcomes [28]. Fundamentally, any change, in the expression of a gene without variation in its nucleotide (DNA) sequence, unlike genetic variations, is known as epigenetic variation. Subsequently, epigenetic studies in diabetic complications may help us to understand the role of epigenetic mechanisms in the alteration of expressions of genes involved in various complications. Hypermethylation at CpG Island in promoter region of a gene is likely to silence its expressions. In contrast, when CpG turns out to be hypomethylated, reverse can takes place [29]. At first, 'epigenetics' term was described by Waddington as 'the casual interaction between genes and their products which bring the phenotype into being' [30]. Epigenetic mechanisms maintains the structure of chromatin to confer transcription memory important for the faithful transmission of gene expression pattern across multiple cell divisions even in the absence of signals that initiated them [31]. Such a control of gene expression by the epigenetic modifications elucidates the mechanisms which triggers our cells with

**3. Epigenetics-an addition to current treatment strategy**

been elaborated in the complications of diabetes.

*DOI: http://dx.doi.org/10.5772/intechopen.94642*

*stimulatory factors.*

complications.

*Diabetes Microvascular Complications: An Overview of Epigenetic Modifications DOI: http://dx.doi.org/10.5772/intechopen.94642*

*Diabetes-induced hyperglycemia promotes various growth factors which play influential roles in the progression of diabetic complications. These factors act by binding to their specific receptors to initiate multiple downstream signaling cascades involved. Subsequently, these signaling pathways trigger transcription factors and promote their crosstalk with epigenetic mechanisms that lead to diabetic microvascular complications. Transcription factors also interact with epigenetic factors that further participate in metabolic memory. AT1R, Angiotensin II type 1 receptor; AGEs, Advance glycation end products; TGF-β, Transforming growth factor-β1; ROS, Reactive oxygen species; NO, Nitric oxide; Akt, Serine/threonine-specific protein kinase; PKC, Protein kinase C; MAPK, Mitogen-activated protein kinase; NF-κB, Nuclear factor-κB; USFs, Upstream stimulatory factors.*

Several genetic factors and gene polymorphisms have been extensively discovered, studied and implicated in DN as well as DR, but no report is able to provide strong evidence regarding uneven response to available treatment. No drug or treatment is able to provide a stable and long-term protective effect against these complications.

In past few years, a lot of interest has been generated in gene–environment interactions as they seem to be involved in the pathophysiology of diabetes mellitus. Recently, epigenetic mechanisms have been linked to various complications of diabetes, as altered gene expressions are the results of several post-transcriptional modifications (PTMs) of chromatin. Accomplishing complete control of blood glucose is also not sufficient to stop or retard the development and progression of diabetes complications; this proposes the involvement of initial glycemic 'metabolic memory' in various complications of diabetes. So, how epigenetic modifications play the role in diabetic vascular complications is still not completely understood, therefore, we have described the understanding about epigenetic mechanisms and their role in the pathophysiology of diabetic microvascular complications.

#### **3. Epigenetics-an addition to current treatment strategy**

In mammalian cells, expression of a gene is known to be controlled by genetic as well as epigenetic mechanisms. In the recent times, epigenetic mechanisms have been shown to play substantial roles in the development and progression of diabetes and its microvascular complications. In this section, epigenetic mechanisms have been elaborated in the complications of diabetes.

Epigenetic mechanisms are deprived of any modification in the principal DNA structure which involves vibrant switching within 'active' (euchromatin) and 'inactive' (heterochromatin) positions of chromatin; that determined 'gene activation' and 'gene repression' states and thus biological outcomes [28]. Fundamentally, any change, in the expression of a gene without variation in its nucleotide (DNA) sequence, unlike genetic variations, is known as epigenetic variation. Subsequently, epigenetic studies in diabetic complications may help us to understand the role of epigenetic mechanisms in the alteration of expressions of genes involved in various complications. Hypermethylation at CpG Island in promoter region of a gene is likely to silence its expressions. In contrast, when CpG turns out to be hypomethylated, reverse can takes place [29]. At first, 'epigenetics' term was described by Waddington as 'the casual interaction between genes and their products which bring the phenotype into being' [30]. Epigenetic mechanisms maintains the structure of chromatin to confer transcription memory important for the faithful transmission of gene expression pattern across multiple cell divisions even in the absence of signals that initiated them [31]. Such a control of gene expression by the epigenetic modifications elucidates the mechanisms which triggers our cells with

*Type 2 Diabetes - From Pathophysiology to Cyber Systems*

DR in Indian population [27].

**Figure 1**.

be reduced by early detection, but it is difficult to achieve as there is little or no symptoms at early stages. Several molecular mechanisms are thought to involve in the development and progression of DR including polyol pathway, enhanced expression of vascular endothelial growth factor (VEGF), production of advance glycation end products (AGEs), activation of RAAS, hemodynamic alterations, etc. Current treatment involves conventional laser therapy and anti-VEGF or other antiangiogenic, anti-inflammatory, non-steroidal anti-inflammatory drugs (NSAIDs) treatment. Despite this, reading is also difficult in patients with severe retina loss. Some treatments are precise but they are associated with high cost or side effects. Hence, the discovery of fundamental molecular mechanisms involved is required for the development of more specific interventions. Among genetic predisposition to the disease, several candidate genes have been identified in the past few decades with contradictory findings, although few genes have been found to be associated with DR in mostly studies. Among them, aldose reductase (AKR1B1) is important enzyme in polyol pathway. Activation of this pathway and AKR1B1 polymorphisms are incriminated in the pathogenesis of DR [24]. VEGF is another the most important growth factor activated by hyperglycemia and implicated in the development and progression of DR [25]. Various polymorphisms of VEGF were found to be associated with DR with conflicting results [24]. ACE I/D polymorphism was found to be associated with PDR in a meta-analysis [26]. Receptor for advance glycation end products (RAGE) gene polymorphisms is also reported to be associated with

The pathophysiology of complications of diabetes is very complex as depicted in

*Signaling pathways facilitating the pathogenesis of microvascular complications of diabetes mellitus.*

**174**

**Figure 1.**

the same DNA to differentiate into numerous cell types with various phenotypes [32, 33]. That's why phenotype of a person is not only decided by its genome but by its epigenome too.

#### **3.1 Factors associated with epigenetic mechanisms**

The suggested mechanism behind altered expression of a gene was the activation of an intracellular signal by environmental factors, which sequentially specifies the accurate chromatin position for epigenetic alterations [34, 35]. Certain environmental aspects takes place during the course of formation and development of embryo (such as maternal diet and intrauterine nutrition) and such an initial development could influence health and disease conditions even at later stages [36]. Additionally, several other environmental exposures accelerate alterations in epigenetic mechanisms, such as heavy metal exposure, smoking, revelation of pesticides, even insufficiencies of nutrients (such as folate and methionine) [37]. Moreover these mechanisms are also appeared to be altered by age and obesity which may possibly cause type 2 DM [38].

#### **3.2 Epigenetic mechanisms in diabetes mellitus and its complications**

In past few years, environment has shown a significant role in activating diabetes, although diabetes has a trend to run in family due to intense genetic component. Obesity, older age and sluggish routine with absence of physical doings are the pronounced risk factors for getting hyperglycemia. Diabetes may cause altered epigenetic mechanisms which can direct diabetes-associated complications such as diabetic nephropathy, by altered expression of genes in target cells as depicted in **Figure 2** [39].

*Diabetes mellitus results in the activation of several signaling pathways following activation of alterations in DNA and histone proteins and transcription factors including NF-κB. These mechanisms via chromatin remodeling resulted in regulated target genes expression in targeted tissues along with activation of several ncRNAs including miRNAs, lncRNAs and circRNAs. Such post transcriptional alterations in target tissues resulted in specific key pathological changes in specific tissues and promote the development of specific vascular complication of diabetes. Even after blood glucose control, synchronized crosstalk between various transcription factors and altered epigenetic mechanisms contribute to the metabolic memory and increased expression of ncRNAs that is embroiled in risk of development of microvascular complication of diabetes.*

High glucose can also stimulate abnormalities in DNA at key genes which are well-known to be involved in endothelial dysfunction as evident by sequencing studies in endothelial cells [40]. Augmented DNA methylation at the promoter region of peroxisome proliferator activated receptor gamma coactivator-1 alpha (PPARGC1A) was reported to be associated with decreased expression of PPARGC1A in pancreatic islets [41]. Tewari et al. [42] have demonstrated the decreased transcriptional activity owing to decreased binding of mitochondrial DNA (mtDNA) to DNA polymerase as a result of hypermethylation at regulatory region of DNA polymerase. Global DNA hypomethylation and thereby, anomalous gene expression due to hyperglycemia was observed in the animal model of diabetes, which was further correlated with inadequate wound healing process [43]. Glucose-induced insulin secretion was shown to be influenced by hyper-acetylation of H4 (histone) at promoter region of insulin gene [38]. Hyperglycemic environment exposure to endothelial cells showed the increased expression of p65 subunit of NF-κB along with other inflammatory genes that correspond with increased H3K4me1 alterations on promoter region of p65 subunit [44].

**177**

as below:

**Figure 2.**

*Diabetes Microvascular Complications: An Overview of Epigenetic Modifications*

Hyperglycemia has also shown to alter micro RNA (miRNA), a mechanism of epigenetic modifications, which is also implicated in complications of diabetes. The alteration in miRNA-133a has been reported in cardiomyocyte hypertrophy in diabetes patients [45]. miRNA-320 upregulation was also observed in myocardial microvascular endothelial cells in rat model with type 2 diabetes [46]. The elementary epigenetic modifications viz., (a) methylation of promoter sites in DNA, (b) modifications in histone proteins and, (c) non-coding RNAs facilitated pathways, as illustrated in **Figure 3**, known to modify the expressions of a gene are described

*Pathways preceding the development of microvascular complications of diabetes.*

*DNA in chromosomes is packed round the histones to form nucleosomes. Unwrapping* 

*and accessibility of nucleosomes is regulated by alterations in histone proteins. DNA methylation involves addition or removal of methyl groups to cytosine residues in CpG islands via DNA methylating enzymes (DNMT) or DNA demethylases, thus, preventing the binding of transcription factors and suppressing respective gene expression. Histone modifications include acetylation, methylation and phosphorylation. HATs/ HDACs regulates the acetylation and deacetylation of histone tails, whereas histone methylation is regulated by HMTs/HDMs. Alterations in histone tail coupled with DNA methylation and control the chromatin accessibility or inaccessibility, hence, regulating the expression of various genes. ncRNAs can be act as housekeeping molecules or regulatory molecules. miRNAs act as regulatory molecules among epigenetic mechanisms and are most widely studied mechanism regulating gene expressions at post-transcriptional level. This dynamic condition of chromatin is exposed to modifications by external* 

*DOI: http://dx.doi.org/10.5772/intechopen.94642*

*Diabetes Microvascular Complications: An Overview of Epigenetic Modifications DOI: http://dx.doi.org/10.5772/intechopen.94642*

#### **Figure 2.**

*Type 2 Diabetes - From Pathophysiology to Cyber Systems*

**3.1 Factors associated with epigenetic mechanisms**

which may possibly cause type 2 DM [38].

its epigenome too.

**Figure 2** [39].

the same DNA to differentiate into numerous cell types with various phenotypes [32, 33]. That's why phenotype of a person is not only decided by its genome but by

of an intracellular signal by environmental factors, which sequentially specifies the accurate chromatin position for epigenetic alterations [34, 35]. Certain environmental aspects takes place during the course of formation and development of embryo (such as maternal diet and intrauterine nutrition) and such an initial development could influence health and disease conditions even at later stages [36]. Additionally, several other environmental exposures accelerate alterations in epigenetic mechanisms, such as heavy metal exposure, smoking, revelation of pesticides, even insufficiencies of nutrients (such as folate and methionine) [37]. Moreover these mechanisms are also appeared to be altered by age and obesity

**3.2 Epigenetic mechanisms in diabetes mellitus and its complications**

H3K4me1 alterations on promoter region of p65 subunit [44].

In past few years, environment has shown a significant role in activating diabetes, although diabetes has a trend to run in family due to intense genetic component. Obesity, older age and sluggish routine with absence of physical doings are the pronounced risk factors for getting hyperglycemia. Diabetes may cause altered epigenetic mechanisms which can direct diabetes-associated complications such as diabetic nephropathy, by altered expression of genes in target cells as depicted in

*Diabetes mellitus results in the activation of several signaling pathways following activation of alterations in DNA and histone proteins and transcription factors including NF-κB. These mechanisms via chromatin remodeling resulted in regulated target genes expression in targeted tissues along with activation of several ncRNAs including miRNAs, lncRNAs and circRNAs. Such post transcriptional alterations in target tissues resulted in specific key pathological changes in specific tissues and promote the development of specific vascular complication of diabetes. Even after blood glucose control, synchronized crosstalk between various transcription factors and altered epigenetic mechanisms contribute to the metabolic memory and increased expression of ncRNAs that is embroiled in risk of development of microvascular complication of diabetes.* High glucose can also stimulate abnormalities in DNA at key genes which are well-known to be involved in endothelial dysfunction as evident by sequencing studies in endothelial cells [40]. Augmented DNA methylation at the promoter region of peroxisome proliferator activated receptor gamma coactivator-1 alpha (PPARGC1A) was reported to be associated with decreased expression of PPARGC1A in pancreatic islets [41]. Tewari et al. [42] have demonstrated the decreased transcriptional activity owing to decreased binding of mitochondrial DNA (mtDNA) to DNA polymerase as a result of hypermethylation at regulatory region of DNA polymerase. Global DNA hypomethylation and thereby, anomalous gene expression due to hyperglycemia was observed in the animal model of diabetes, which was further correlated with inadequate wound healing process [43]. Glucose-induced insulin secretion was shown to be influenced by hyper-acetylation of H4 (histone) at promoter region of insulin gene [38]. Hyperglycemic environment exposure to endothelial cells showed the increased expression of p65 subunit of NF-κB along with other inflammatory genes that correspond with increased

The suggested mechanism behind altered expression of a gene was the activation

**176**

*Pathways preceding the development of microvascular complications of diabetes.*

Hyperglycemia has also shown to alter micro RNA (miRNA), a mechanism of epigenetic modifications, which is also implicated in complications of diabetes. The alteration in miRNA-133a has been reported in cardiomyocyte hypertrophy in diabetes patients [45]. miRNA-320 upregulation was also observed in myocardial microvascular endothelial cells in rat model with type 2 diabetes [46]. The elementary epigenetic modifications viz., (a) methylation of promoter sites in DNA, (b) modifications in histone proteins and, (c) non-coding RNAs facilitated pathways, as illustrated in **Figure 3**, known to modify the expressions of a gene are described as below:

*DNA in chromosomes is packed round the histones to form nucleosomes. Unwrapping and accessibility of nucleosomes is regulated by alterations in histone proteins. DNA methylation involves addition or removal of methyl groups to cytosine residues in CpG islands via DNA methylating enzymes (DNMT) or DNA demethylases, thus, preventing the binding of transcription factors and suppressing respective gene expression. Histone modifications include acetylation, methylation and phosphorylation. HATs/ HDACs regulates the acetylation and deacetylation of histone tails, whereas histone methylation is regulated by HMTs/HDMs. Alterations in histone tail coupled with DNA methylation and control the chromatin accessibility or inaccessibility, hence, regulating the expression of various genes. ncRNAs can be act as housekeeping molecules or regulatory molecules. miRNAs act as regulatory molecules among epigenetic mechanisms and are most widely studied mechanism regulating gene expressions at post-transcriptional level. This dynamic condition of chromatin is exposed to modifications by external* 

**Figure 3.** *Framework of inheritable epigenetic modifications.*

*stimuli via regulation of miRNAs, thus directing several pathophysiological outcomes. DNMTs, DNA methyl transferases; HATs, Histone acetyl transferases; HDACs, Histone deacetylases; HMTs, Histone methyl transferases; HDMs, Histone demethylases; ncRNAs, non-coding RNAs, miRNAs, micro RNAs.*
